Branched-chain amino acids (BCAAs), namely leucine, isoleucine and valine, are synthesized by plants, algae, fungi, bacteria and archaea, but not by animals. BCCAs are essential amino acids for humans and are used clinically for the treatment of burns and hepatic encephalopathy as well as for increasing muscle mass. The enzymes of the BCAA biosynthetic pathway are potential targets for the development of herbicides, fungicides, and antimicrobial compounds. Some of the most popular herbicides (e.g. sulfometuron methyl, SMM), act by inhibiting the first common enzyme in the BCAA biosynthetic pathway, acetohydroxyacid synthase (AHAS). Since AHAS is required for the synthesis of all three BCAAs, its inhibition is detrimental to the organism and effectively inhibits its growth. Plant and green algal AHAS are localized in the chloroplast and fungal AHAS in the mitochondria, although the genes may be present in the nuclear or organelle genome. In cases of nuclear-encoded genes, the enzyme is transported to the target subcellullar compartment by an additional, poorly conserved, N-terminal targeting peptide.
Microalgae are one of the richest sources of long-chain polyunsaturated fatty acids (LC-PUFAs). The green freshwater microalga Parietochloris incisa (Trebouxiophyceae) is the only microalga able to accumulate extraordinary high amounts of LC- PUFA arachidonic acid (ARA)-rich triacylglycerols (TAG). When P. incisa is cultivated under nitrogen starvation, the fatty acid (FA) content of the alga is over 35% of dry weight; ARA constitutes about 60% of total FAs, and over 90% of cell ARA is deposited in TAG, making it the richest green dietary source of ARA. LC-PUFAs include the ω3-fatty acids, eicosapentaenoic acid (EPA, 20:5ω3), docosahexaenoic acid (DHA, 22:6ω3), ω6-fatty acid, arachidonic acid (ARA, 20:4ω6) and dihomo-γ-linolenic acid (DGLA, 20:3ω6). LC-PUFA are major components of membrane phospholipids of the retina, brain and testis and are predominant in the human brain and breast milk (specifically ARA and DHA). ARA is necessary for normal fetal growth and for cognitive development in infants and is also the primary substrate in eicosanoids biosynthesis, which regulates many physiological processes such as homeostasis, reproduction, immune and inflammatory responses.
Use of antibiotic resistance genes as selection markers in transformation processes presents numerous environmental and health risks, as well as regulatory difficulties that define the organism as genetically modified (GM). Thus, a selection system based on mutation(s) of an endogenous gene(s) is highly advantageous. It has been reported that mutant forms of AHAS exhibit herbicide resistance in yeast, higher plants and green algae, where most of the characterized AHAS herbicide resistances is due to a single or double amino acid change from the wild-type enzyme sequence. At least 17 different amino acid substitutions in AHAS are known to confer resistance to growth inhibiting herbicides. For example, in tobacco, a resistant mutant with a single amino acid change of Tryptophan557, within a conserved region of AHAS, was found to be insensitive to inhibition by two sulfonylurea herbicides, chlorsulfuron and SMM. In addition, corresponding Trp residue mutations were shown to be important for AHAS SMM resistance of Escherichia coli, Mycobacterium tuberculosis, Brassica napus and the red microalga Porphyridium sp.
There remains an unmet need for the development of cells, in particular microalgal cells which are capable of producing LC-PUFA in large scale systems and which are resistant to herbicides, using methods which do not classify the organism as genetically modified.